PROJECT SUMMARY/ABSTRACT Cells in the osteoblast lineage are crucial for controlling bone formation and bone resorption throughout life. An in-depth understanding of how signaling processes within osteoblast-lineage cells are linked to bone formation and bone resorption is essential to provide new insights into skeletal physiology and to open up new avenues for osteoporosis therapy. In this regard, the G protein signaling pathway is crucial in that the only FDA-approved anabolic treatment for osteoporosis is intermittent parathyroid hormone which acts through a G protein coupled receptor (GPCR) in osteoblasts. It is thus vital that we increase our understanding of how specific G protein signals in osteoblasts are linked to bone formation and bone resorption, and when during the process of osteoblastogenesis these signals act. The complexity of the skeletal environment for normal bone formation and turnover make it necessary to use in vivo models to address these critical issues. During the previous grant period, our laboratory has established the utility of an approach to dissect the role of specific signaling pathways using transgenic mice expressing engineered GPCRs termed RASSLs (Receptors Activated Solely by Synthetic Ligands). Our results so far demonstrate that activation of the Gi signaling pathway in mature osteoblasts results in trabecular osteopenia due to decreased rates of bone formation. By contrast, activation of the Gs signaling pathway in mature osteoblasts results in a massive and disorganized increase in the formation of trabecular bone with a marked reduction in marrow elements and erosion of the cortex. In the present proposal, we will extend these observations and probe their pathophysiological relevance. Specifically, we propose to: 1) assess the role of an endogenous, constitutively active Gi-couple GPCR (EBI2) in regulating skeletal homeostasis, and determine whether increased Gi signaling contributes to the pathogenesis of bone loss in mice following ovariectomy;2) determine the role of Gq signaling in mature osteoblasts and assess whether coordinate activation of Gs and Gq signaling is required for normal skeletal anabolism;and 3) evaluate the potential of Gs signaling in immature osteoblasts to normalize the disordered anabolic response to Gs signaling in mature osteoblasts. The knowledge gained from these studies will provide new insights into the control of osteoblastic bone formation, and these will be valuable in the design and development of improved anabolic therapies for osteoporosis.